The main obstacle to harnessing the power of RNA interference for disease prevention is delivering small RNAs across the cell plasma membrane in vivo. This is an especially formidable obstacle for T lymphocytes, which are a moving target in the body and are refractory to transduction by lipid transfection agents. We have engineered, in vitro transcribed, purified and tested chimeric small RNAs designed to deliver small interfering RNAs (siRNAs) into human CD4+ cells. These chimeras are composed of a structured piece of RNA, called an aptamer, that recognizes and binds to CD4, which is linked at its 3'-end to the sense (or inactive passenger) strand of an siRNA. This strand of the RNA is then annealed to a small antisense (active strand) RNA. The CD4 aptamer-siRNA chimeras specifically enter and induce gene silencing in CD4+ cells, but not in CD8 T cells. Chimeras silence gene expression both in CD4+ cell lines (HeLa-CD4, Jurkat) and in primary CD4+ human resting or activated PBMCs. Moreover, pretreatment of primary T cells and macrophages, as well as polarized cervicovaginal tissue explants, with CD4 aptamer-siRNAs directed against CCR5 and/or HIV genes inhibits HIV replication. Therefore the chimeras traffic within tissues to find and transduce immune cells within the lamina propria in intact tissues. The aptamer-siRNA chimeras do not damage the genital mucosa or induce expression of IFNs, IFN response genes, IL-12, IL-6 or IL-8 in tissues. Preliminary data obtained after intravaginal application of CD4 aptamer-siRNA chimeras in NOD/scid/IL2R?-/- mice reconstituted with human bone marrow, fetal liver and thymus (BLT mice) show both uptake of Cy3-labeled chimeras and CCR5 silencing in cervicovaginal tissue CD4+ cells. Based on these preliminary results, we hypothesize that intravaginal application of CD4 aptamer-siRNA chimeras targeting HIV and/or CCR5 can prevent sexual transmission of HIV without causing undue toxicity. Moreover we expect that chimeric RNAs are practical as the active ingredient in a microbicide suitable for global use and that protection will last for about a week implying that topical application will not need to be performed just before intercourse. This proposal will begin to evaluate whether CD4 aptamer-siRNA chimeras can be used as the active component of a topical microbicide to prevent sexual transmission of HIV. To accomplish this goal, we will (1) test the utility of siRNA- CD4 aptamers in vitro and in polarized explants for cell-specific uptake, gene silencing, inhibition of HIV infection, and potential toxicity; (2) optimize the design of siRNA-CD4 aptamers; and (3) test siRNA-CD4 aptamers administered intravaginally to humanized BLT mice for their effectiveness and durability of protection from HIV vaginal transmission. In the absence of an effective vaccine, topical microbicides could provide a way to help control the global HIV pandemic. An siRNA-based microbicide, an approach which has been shown to inhibit HSV-2 transmission for at least a week in mice, provides a method to establish relatively long-lived protection against sexual transmission in the genital tract to obviate the need for treatment just before sexual intercourse. We propose to investigate in this proposal a novel method for overcoming the major obstacle to extending the promising results for inhibiting HSV-2 transmission to HIV, namely getting small RNAs into the immune cells that HIV infects.